New perspectives on the melanocortins and their cardiovascular effects: Potential implications for the treatment of cardiovascular diseases with melanocortin analogues

The melanocortin peptides, including melanocyte-stimulating hormones, α-, β- and γ-MSH, are derived from the precursor peptide proopiomelanocortin and mediate their biological actions via five different melanocortin receptors, named from MC1 to MC5. Melanocortins have been implicated in the central regulation of energy balance and cardiovascular functions, but their local effects, via yet unidentified sites of action, in the vasculature, and their therapeutic potential in major vascular pathologies remain unclear. Therefore, the main aim of this thesis was to characterise the role of melanocortins in circulatory regulation, and to investigate whether targeting of the melanocortin system by pharmacological means could translate into therapeutic benefits in the treatment of cardiovascular diseases such as hypertension. In experiments designed to elucidate the local effects of α-MSH on vascular tone, it was found that α-MSH improved blood vessel relaxation via a nitric oxide (NO)-dependent mechanism without directly contracting or relaxing blood vessels. Furthermore, α-MSH was shown to regulate the expression and function of endothelial NO synthase in cultured human endothelial cells via melanocortin 1 receptors. In keeping with the vascular protective role, pharmacological treatment of mice with α-MSH analogues displayed therapeutic efficacy in conditions associated with vascular dysfunction such as obesity. Furthermore, α-MSH analogues elicited marked diuretic and natriuretic responses, which together with their vascular effects, seemed to provide protection against sodium retention and blood pressure elevation in experimental models of hypertension. In conclusion, the present results identify novel effects for melanocortins in the local control of vascular function, pointing to the potential future use of melanocortin analogues in the treatment of cardiovascular pathologies.

Identification of Epigenetic Targets in Prostate Cancer for Therapeutic Development

Recurrent castration resistant prostate cancer remains a challenge for cancer therapies and novel treatment options in addition to current anti-androgen and mitosis inhibitors are needed. Aberrations in epigenetic enzymes and chromatin binding proteins have been linked to prostate cancer and they may form a novel class of drug targets in the future. In this thesis we systematically evaluated the epigenenome as a prostate cancer drug target. We functionally silenced 615 known and putative epigenetically active protein coding genes in prostate cancer cell lines using high throughput RNAi screening and evaluated the effects on cell proliferation, androgen receptor (AR) expression and histone patterns. Histone deacetylases (HDACs) were found to regulate AR expression. Furthermore, HDAC inhibitors reduced AR signaling and inhibited synergistically with androgen deprivation prostate cancer cell proliferation. In particular, TMPRSS2- EGR fusion gene positive prostate cancer cell lines were sensitive to combined HDAC and AR inhibition, which may partly be related to the dependency of a fusion gene induced epigenetic pathway. Histone demethylases (HDMs) were identified to regulate prostate cancer cell line proliferation. We discovered a novel histone JmjC-domain histone demethylase PHF8 to be highly expressed in high grade prostate cancers and mediate cell proliferation, migration and invasion in in vitro models. Additionally, we explored novel HDM inhibitor chemical structures using virtual screening methods. The structures best fitting to the active pocket of KDM4A were tested for enzyme inhibition and prostate cancer cell proliferation activity in vitro. In conclusion, our results show that prostate cancer may efficiently be targeted with combined AR and HDAC inhibition which is also currently being tested in clinical trials. HDMs were identified as another feasible novel drug target class. Future studies in representative animal models and development of specific inhibitors may reveal HDMs full potential in prostate cancer therapy

The Effects of Fibroblast Growth Factor 8b on Reproductive Organs and Prostate Tumorigenesis

Fibroblast growth factors (FGFs) are involved in the development and homeostasis of the prostate and other reproductive organs. FGF signaling is altered in prostate cancer. Fibroblast growth factor 8 (FGF8) is a mitogenic growth factor and its expression is elevated in prostate cancer and in premalignant prostatic intraepithelial neoplasia (PIN) lesions. FGF8b is the most transforming isoform of FGF8. Experimental models show that FGF8b promotes several phases of prostate tumorigenesis - including cancer initiation, tumor growth, angiogenesis, invasion and development of bone metastasis. The mechanisms activated by FGF8b in the prostate are unclear. In the present study, to examine the tumorigenic effects of FGF8b on the prostate and other FGF8b expressing organs, an FGF8b transgenic (TG) mouse model was generated. The effect of estrogen receptor beta (ERβ) deficiency on FGF8binduced prostate tumorigenesis was studied by breeding FGF8b-TG mice with ERβ knockout mice (BERKOFVB). Overexpression of FGF8b caused progressive histological and morphological changes in the prostate, epididymis and testis of FGF8b-TG-mice. In the prostate, hyperplastic, preneoplastic and neoplastic changes, including mouse PIN (mPIN) lesions, adenocarcinomas, sarcomas and carcinosarcomas were present in the epithelium and stroma. In the epididymis, a highly cancer-resistant tissue, the epithelium contained dysplasias and the stroma had neoplasias and hyperplasias with atypical cells. Besides similar histological changes in the prostate and epididymis, overexpression of FGF8b induced similar changes in the expression of genes such as osteopontin (Spp1), connective tissue growth factor (Ctgf) and FGF receptors (Fgfrs) in these two tissues. In the testes of the FGF8b-TG mice, the seminiferous epithelium was frequently degenerative and the number of spermatids was decreased. A portion of the FGF8b-TG male mice was infertile. Deficiency of ERβ did not accelerate prostate tumorigenesis in the FGF8b-TG mice, but increased significantly the frequency of mucinous metaplasia and slightly the frequency of inflammation in the prostate. This suggests putative differentiation promoting and anti-inflammatory roles for ERβ. In summary, these results underscore the importance of FGF signaling in male reproductive organs and provide novel evidence for a role of FGF8b in stromal activation and prostate tumorigenesis.

Markers of Bone Turnover In Preclinical Development of Drugs for Skeletal Diseases

Skeletal tissue is constantly remodeled in a process where osteoclasts resorb old bone and osteoblasts form new bone. Balance in bone remodeling is related to age, gender and genetic factors, but also many skeletal diseases, such as osteoporosis and cancer-induced bone metastasis, cause imbalance in bone turnover and lead to decreased bone mass and increased fracture risk. Biochemical markers of bone turnover are surrogates for bone metabolism and may be used as indicators of the balance between bone resorption and formation. They are released during the remodeling process and can be conveniently and reliably measured from blood or urine by immunoassays. Most commonly used bone formation markers include N-terminal propeptides of type I collagen (PINP) and osteocalcin, whereas tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) and C-terminal cross-linked telopeptide of type I collagen (CTX) are common resorption markers. Of these, PINP has been, until recently, the only marker not commercially available for preclinical use. To date, widespread use of bone markers is still limited due to their unclear biological significance, variability, and insufficient evidence of their prognostic value to reflect long term changes. In this study, the feasibility of bone markers as predictors of drug efficacy in preclinical osteoporosis models was elucidated. A non-radioactive PINP immunoassay for preclinical use was characterized and validated. The levels of PINP, N-terminal mid-fragment of osteocalcin, TRACP 5b and CTX were studied in preclinical osteoporosis models and the results were compared with the results obtained by traditional analysis methods such as histology, densitometry and microscopy. Changes in all bone markers at early timepoints correlated strongly with the changes observed in bone mass and bone quality parameters at the end of the study. TRACP 5b correlated strongly with the osteoclast number and CTX correlated with the osteoclast activity in both in vitro and in vivo studies. The concept “resorption index” was applied to the relation of CTX/TRACP 5b to describe the mean osteoclast activity. The index showed more substantial changes than either of the markers alone in the preclinical osteoporosis models used in this study. PINP was strongly associated with bone formation whereas osteocalcin was associated with both bone formation and resorption. These results provide novel insight into the feasibility of PINP, osteocalcin, TRACP 5b and CTX as predictors of drug efficacy in preclinical osteoporosis models. The results support clinical findings which indicate that short-term changes of these markers reflect long-term responses in bone mass and quality. Furthermore, this information may be useful when considering cost-efficient and clinically predictive drug screening and development assays for mining new drug candidates for skeletal diseases.

Quantitative Analysis of Novel Prostate Cancer Markers in Tissue

Prostate cancer is a heterogeneous disease affecting an increasing number of men all over the world, but particularly in the countries with the Western lifestyle. The best biomarker assay currently available for the diagnosis of the disease, the measurement of prostate specific antigen (PSA) levels from blood, lacks specificity, and even when combined with invasive tests such as digital rectal exam and prostate tissue biopsies, these methods can both miss cancers, and lead to overdiagnosis and subsequent overtreatment of cancers. Moreover, they cannot provide an accurate prognosis for the disease. Due to the high prevalence of indolent prostate cancers, the majority of men affected by prostate cancer would be able to live without any medical intervention. Their latent prostate tumors would not cause any clinical symptoms during their lifetime, but few are willing to take the risk, as currently there are no methods or biomarkers to reliably differentiate the indolent cancers from the aggressive, lethal cases that really are in need of immediate medical treatment. This doctoral work concentrated on validating 12 novel candidate genes for use as biomarkers for prostate cancer by measuring their mRNA expression levels in prostate tissue and peripheral blood of men with cancer as well as unaffected individuals. The panel of genes included the most prominent markers in the current literature: PCA3 and the fusion gene TMPRSS2-ERG, in addition to BMP-6, FGF-8b, MSMB, PSCA, SPINK1, and TRPM8; and the kallikrein-related peptidase genes 2, 3, 4, and 15. Truly quantitative reverse-transcription PCR assays were developed for each of the genes for the purpose, time-resolved fluorometry was applied in the real-time detection of the amplification products, and the gene expression data were normalized by using artificial internal RNA standards. Cancer-related, statistically significant differences in gene transcript levels were found for TMPRSS2-ERG, PCA3, and in a more modest scale, for KLK15, PSCA, and SPINK1. PCA3 RNA was found in the blood of men with metastatic prostate cancer, but not in localized cases of cancer, suggesting limitations for using this method for early cancer detection in blood. TMPRSS2-ERG mRNA transcripts were found more frequently in cancerous than in benign prostate tissues, but they were present also in 51% of the histologically benign prostate tissues of men with prostate cancer, while being absent in specimens from men without any signs of prostate cancer. PCA3 was shown to be 5.8 times overexpressed in cancerous tissue, but similarly to the fusion gene mRNA, its levels were upregulated also in the histologically benign regions of the tissue if the corresponding prostate was harboring carcinoma. These results indicate a possibility to utilize these molecular assays to assist in prostate cancer risk evaluation especially in men with initially histologically negative biopsies.

Wood biochemicals for the protection of health. Focus on hemicellulose, stilbenoids and lignans

Trees produce an enormous amount of compounds that are still scantly utilized.However, the results obtained from structurally similar biochemicals suggest that wood-derived compounds could be used for the protection of health in various applications. Polyphenols, for instance, could be extracted from wood in high quantities. Similar polyphenols to those in wood include resveratrol, found in grapes, and secoisolariciresinol, present in flaxseeds. Their consumption has been inversely associated with the incidence of various diseases, especially certain cancers and obesity-related disorders. The aim of this study was to determine the health-promoting effects of woodderived biochemicals. The effect of spruce hemicellulose on the growth of probiotic intestinal bacteria was studied. The results suggest that the bifidobacteria and lactobacilli can utilize hemicellulose and thus it has potential as a prebiotic compound. In particular, the efficacy of pine polyphenols to inhibit the growth of prostate cancer was our main interest. It was found that stilbenoids and lignans inhibited the proliferation of various cancer cells, and reduced the growth of prostate cancer xenografts in mice. The polyphenol rich pine knot extract was well tolerated in diet and extract-derived polyphenols were rapidly absorbed after intake. Furthermore, we determined the effect of the dietary pine knot extract on the weight gain and the expression of aromatase gene in reporter mouse expressing the promoter region of a human aromatase gene. It was found that dietary pine knot extract alleviated the obesity-induced inflammation in adipose tissue and downregulated the expression of a human aromatase gene. Taken together, several components of spruce and pine may have a future role as health-promoting compounds.

To divide or differentiate? : nestin-mediated regulation of Cdk5 in cell fate decisions

The molecular functions of the non-cell cycle-related Cyclin-dependent kinase 5 (Cdk5) have been of primary interest within the neuroscience field, but novel undertakings are constantly emerging for the kinase in tissue homeostasis, as well as in diseases such as diabetes and cancer. Although Cdk5 activation is predominantly regulated by specific non-cyclin activator protein binding, additional mechanisms have proved to orchestrate Cdk5 signaling in cells. For example, the interaction between the intermediate filament protein nestin and Cdk5 has been proposed to determine cellular fate during neuronal apoptosis through nestin-dependent adjustment of the sensitive balance and turnover of Cdk5 activators. While nestin constitutes a crucial regulatory scaffold for appropriate Cdk5 activation in apoptosis, Cdk5 itself phosphorylates nestin with the consequence of filament reorganization in both neuronal progenitors and differentiating muscle cells. Interestingly, the two proteins are often found coexpressed in various tissues and cell types, proposing that nestin-mediated scaffolding of Cdk5 and its activators may be applicable to other tissue systems as well. In the literature, the molecular functions of nestin have remained in the shade, as it is mostly exploited as a marker protein for progenitor cells. In light of these studies, the aim of this thesis was to assess the importance of the nestin scaffold in regulation of Cdk5 actions in cell fate decisions. This thesis can be subdivided into two major projects: one that studied the nature of the Cdk5-nestin interplay in muscle, and one that assessed their role in prostate cancer. During differentiation of a myoblast cell line, the filament formation properties of nestin was found to be crucial in directing Cdk5 activity, with direct consequences on the process of differentiation. Also the genetic knockout of nestin was found to influence Cdk5 activity, although differentiation per se was not affected. Instead, the genetic ablation of nestin had broad consequences on muscle homeostasis and regeneration. While the nestin-mediated regulation of Cdk5 in muscle was found to act in multiple ways, the connection remained more elusive in cancer models. Cdk5 was, however, established as a significant determinant of prostate cancer proliferation; a behavior uncharacteristic for this differentiation-associated kinase. Through complex and simultaneous regulation of two major prostate cancer pathways, Cdk5 was placed upstream of both Akt kinase and the androgen receptor. Its action on proliferation was nonetheless mainly exerted through the Akt signaling pathway in various cancer models. In summary, this thesis contributed to the knowledge of Cdk5 regulation and functions in two atypical settings; proliferation (in a cancer framework) and muscle differentiation, which is a poorly understood model system in the Cdk5 field. This balance between proliferation and differentiation implemented by Cdk5 is ultimately regulated (where present) by the dynamics of the cytoskeletal nestin scaffold.

Scalable Feature Selection Applications for Genome-Wide Association Studies of Complex Diseases

Personalized medicine will revolutionize our capabilities to combat disease. Working toward this goal, a fundamental task is the deciphering of geneticvariants that are predictive of complex diseases. Modern studies, in the formof genome-wide association studies (GWAS) have afforded researchers with the opportunity to reveal new genotype-phenotype relationships through the extensive scanning of genetic variants. These studies typically contain over half a million genetic features for thousands of individuals. Examining this with methods other than univariate statistics is a challenging task requiring advanced algorithms that are scalable to the genome-wide level. In the future, next-generation sequencing studies (NGS) will contain an even larger number of common and rare variants. Machine learning-based feature selection algorithms have been shown to have the ability to effectively create predictive models for various genotype-phenotype relationships. This work explores the problem of selecting genetic variant subsets that are the most predictive of complex disease phenotypes through various feature selection methodologies, including filter, wrapper and embedded algorithms. The examined machine learning algorithms were demonstrated to not only be effective at predicting the disease phenotypes, but also doing so efficiently through the use of computational shortcuts. While much of the work was able to be run on high-end desktops, some work was further extended so that it could be implemented on parallel computers helping to assure that they will also scale to the NGS data sets. Further, these studies analyzed the relationships between various feature selection methods and demonstrated the need for careful testing when selecting an algorithm. It was shown that there is no universally optimal algorithm for variant selection in GWAS, but rather methodologies need to be selected based on the desired outcome, such as the number of features to be included in the prediction model. It was also demonstrated that without proper model validation, for example using nested cross-validation, the models can result in overly-optimistic prediction accuracies and decreased generalization ability. It is through the implementation and application of machine learning methods that one can extract predictive genotype–phenotype relationships and biological insights from genetic data sets.

Genetics of inherited small vessel diseases – in search of a novel small vessel disease and modifiers of the clinical course of CADASIL

The genetic and environmental risk factors of vascular cognitive impairment are still largely unknown. This thesis aimed to assess the genetic background of two clinically similar familial small vessel diseases (SVD), CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) and Swedish hMID (hereditary multi-infarct dementia of Swedish type). In the first study, selected genetic modifiers of CADASIL were studied in a homogenous Finnish CADASIL population of 134 patients, all carrying the p.Arg133Cys mutation in NOTCH3. Apolipoprotein E (APOE) genotypes, angiotensinogen (AGT) p.Met268Thr polymorphism and eight NOTCH3 polymorphisms were studied, but no associations between any particular genetic variant and first-ever stroke or migraine were seen. In the second study, smoking, statin medication and physical activity were suggested to be the most profound environmental differences among the monozygotic twins with CADASIL. Swedish hMID was for long misdiagnosed as CADASIL. In the third study, the CADASIL diagnosis in the Swedish hMID family was ruled out on the basis of genetic, radiological and pathological findings, and Swedish hMID was suggested to represent a novel SVD. In the fourth study, the gene defect of Swedish hMID was then sought using whole exome sequencing paired with a linkage analysis. The strongest candidate for the pathogenic mutation was a 3’UTR variant in the COL4A1 gene, but further studies are needed to confirm its functionality. This study provided new information about the genetic background of two inherited SVDs. Profound knowledge about the pathogenic mutations causing familial SVD is also important for correct diagnosis and treatment options.

Clinical and genetic markers in disease progression – a study among subjects with obstructive lung diseases

Asthma, COPD, and asthma and COPD overlap syndrome (ACOS) are chronic pulmonary diseases with an obstructive component. In COPD, the obstruction is irreversible and the disease is progressive. The aim of the study was to define and analyze factors that affected disease progression and patients’ well-being, prognosis and mortality in Chronic Airway Disease (CAD) cohort. The main focus was on COPD and ACOS patients. Retrospective data from medical records was combined with genetic and prospective follow-up data. Smoking is the biggest risk factor for COPD and even after the diagnosis of the disease, smoking plays an important role in disease development and patient’s prognosis. Sixty percent of the COPD patients had succeeded in smoking cessation. Patients who had managed to quit smoking had lower mortality rates and less psychiatric diseases and alcohol abuse although they were older and had more cardiovascular diseases than patients who continued smoking. Genetic polymorphism rs1051730 in the nicotinic acethylcholine receptor gene (CHRNA3/5) associated with heavy smoking, cancer prevalence and mortality in two Finnish independent cohorts consisting of COPD patients and male smokers. Challenges in smoking cessation and higher mortality rates may be partly due to individual patient’s genetic composition. Approximately 50% of COPD patients are physically inactive and the proportion was higher among current smokers. Physically active and inactive patients didn’t differ from each other in regard to age, gender or comorbidities. Bronchial obstruction explained inactivity only in severe disease. Subjective sensation of dyspnea, however, had very strong association to inactivity and was also associated to low health related quality of life (HRQoL). ACOS patients had a significantly lower HRQoL than either the patients with asthma or with COPD even though they were younger than COPD patients, had better lung functions and smaller tobacco exposure.

Identification and validation of novel prostate cancer biomarkers

Prostate cancer (PCa) has emerged as the most commonly diagnosed lethal cancer in European men. PCa is a heterogeneous cancer that in the majority of the cases is slow growing: consequently, these patients would not need any medical treatment. Currently, the measurement of prostate-specific antigen (PSA) from blood by immunoassay followed by digital rectal examination and a pathological examination of prostate tissue biopsies are the most widely used methods in the diagnosis of PCa. These methods suffer from a lack of sensitivity and specificity that may cause either missed cancers or overtreatment as a consequence of over-diagnosis. Therefore, more reliable biomarkers are needed for a better discrimination between indolent and potentially aggressive cancers. The aim of this thesis was the identification and validation of novel biomarkers for PCa. The mRNA expression level of 14 genes including AMACR, AR, PCA3, SPINK1, TMPRSS2-ERG, KLK3, ACSM1, CACNA1D, DLX1, LMNB1, PLA2G7, RHOU, SPON2, and TDRD1 was measured by a truly quantitative reverse transcription PCR in different prostate tissue samples from men with and without PCa. For the last eight genes the function of the genes in PCa progression was studied by a specific siRNA knockdown in PC-3 and VCaP cells. The results from radical prostatectomy and cystoprostatectomy samples showed statistically significant overexpression for all the target genes, except for KLK3 in men with PCa compared with men without PCa. Statistically significant difference was also observed in low versus high Gleason grade tumors (for PLA2G7), PSA relapse versus no relapse (for SPON2), and low versus high TNM stages (for CACNA1D and DLX1). Functional studies and siRNA silencing results revealed a cytotoxicity effect for the knock-down of DLX1, PLA2G7, and RHOU, and altered tumor cell invasion for PLA2G7, RHOU, ACSM1, and CACNA1D knock-down in 3D conditions. In addition, effects on tumor cell motility were observed after silencing PLA2G7 and RHOU in 2D monolayer cultures. Altogether, these findings indicate the possibility of utilizing these new markers as diagnostic and prognostic markers, and they may also represent therapeutic targets for PCa.